The invention relates to essential bacterial genes and their use in identifying antibacterial agents.
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system. The bacterium Streptococcus pneumonia typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
The invention is based on the discovery of 23 genes in the bacterium Streptococcus pneumoniae, and a related gene in the bacterium Bacillus subtilis, that are located within operons that are essential for survival. These 23 Streptococcus genes are referred to herein as xe2x80x9cGEP genesxe2x80x9d (which stands for general essential protein); for convenience, the polypeptides encoded by these genes are referred to herein as xe2x80x9cGEP polypeptides.xe2x80x9d Each GEP gene is located within an operon that contains a gene that is essential for survival of Streptococcus pneumoniae; the essential gene can be the GEP gene or another gene located within the same operon. Bacterial operons contain several genes that are related, e.g., with respect to function or biochemical pathway. Transcription of an operon leads to the production of a single transcript in which multiple coding regions are linked. Thus, an operon containing one or more essential genes can be considered an xe2x80x9cessential operon,xe2x80x9d since disruption of expression of one gene located within the operon will interfere with expression of the other genes in the operon. Each coding region of the transcript is separately translated into an individual polypeptide by ribosomes that initiate translation at multiple points along the transcript. Having identified one gene in the operon, one can readily identify and sequence the other genes located within the operon.
The genes encoding the GEP polypeptides are useful molecular tools for identifying similar genes in pathogenic microorganisms, such as pathogenic strains of Bacillus. In addition, the operons containing genes encoding GEP polypeptides, and the polypeptides encoded by such operons, are useful targets for identifying compounds that are inhibitors of the pathogens in which the GEP polypeptides are expressed. Such inhibitors inhibit bacterial growth by being bacteriostatic (e.g., inhibiting reproduction or cell division) or by being bacteriocidal (i.e., by causing cell death).
The invention, therefore, features an isolated polypeptide encoded by a nucleic acid located within an operon encoding a GEP polypeptide, termed gep103, having the amino acid sequence set forth in SEQ ID NO:1, or conservative variations thereof. An isolated operon comprising a nucleic acid encoding gep103 also is included within the invention. In addition, the invention includes an isolated nucleic acid of (a) an operon comprising the sequence of SEQ ID NO:2, as depicted in FIG. 1, or degenerate variants thereof; (b) an operon comprising the sequence of SEQ ID NO:2, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:1. As described above for gep103, other nucleic acids and polypeptides encoded by nucleic acids located within operons encoding GEP polypeptides are included within the invention, including: (a) operons comprising the nucleic acids represented by the SEQ ID NOs. listed below, as depicted in the Figures listed below, or degenerate variants thereof; (b) operons comprising the nucleic acids represented by the SEQ ID NOs. listed below, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA 5 encoding the polypeptides represented by the SEQ ID NOs. listed below.
The invention also includes allelic variants (i.e., genes encoding isozymes) of the genes located within operons encoding the GEP polypeptides listed above. For example, the invention includes a gene that encodes a GEP polypeptide but which gene includes one or more point mutations, deletions, promotor variants, or splice site variants, provided that the resulting GEP polypeptide functions as a GEP polypeptide (e.g., as determined in a conventional complementation assay).
Identification of these GEP genes and the determination that they are located within operons containing an essential gene allows homologs of the GEP genes to be found in other organisms strains of Streptococcus. Also, orthologs of these genes can be identified in other species (e.g., Bacillus sp.). While xe2x80x9chomologsxe2x80x9d are structurally similar genes contained within a species, xe2x80x9corthologsxe2x80x9d are functionally equivalent genes from other species (within or outside of a given genus, e.g., from Bacillus subtilis or E. coli). Such homologs and orthologs are expected to be located within operons that are essential for survival. Such homologous and orthologous genes and polypeptides can be used to identify compounds that inhibit the growth of the host organism (e.g., compounds that are bacteriocidal or bacteriostatic against pathogenic strains of the organism). Homologous and orthologous genes and polypeptides that are essential for survival can serve as targets for identifying a broad spectrum of antibacterial agents.
An ortholog of gep1493, termed B-yneS, has been identified in B. subtilis and is essential for survival of B. subtilis. The amino acid sequence (SEQ ID NO: 70), coding sequence (SEQ ID NO:71), and non-coding sequence (SEQ ID NO:72) of B-yneS is set forth in FIGS. 24A-B. As with the other polypeptides and genes disclosed herein, the B-yneS polypeptide and gene can be used in the methods described herein to identify antibacterial agents.
The term gep103 polypeptide or gene as used herein is intended to include the polypeptide and gene set forth in FIG. 1 herein, as well as homologs of the sequences set forth in FIG. 1. Also encompassed by the term gep103 gene are degenerate variants of the nucleic acid sequence set forth in FIG. 1 (SEQ ID NO:2). Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequence represented by SEQ ID NO:2, but which nonetheless encode a gep103 polypeptide are included within the invention. Likewise, because of the similarity in the structures of amino acids, conservative variations (as described herein) can be made in the amino acid sequence of the gep103 polypeptide while retaining the function of the polypeptide (e.g., as determined in a conventional complementation assay). Other gep103 polypeptides and genes identified in additional Streptococcus strains may be such conservative variations or degenerate variants of the particular gep103 polypeptide and nucleic acid set forth in FIG. 1 (SEQ ID NOs:1 and 2, respectively). The gep103 polypeptide and gene share at least 80%, e.g., 90%, sequence identity with SEQ ID NOs:1 and 2, respectively. Regardless of the percent sequence identity between the gep103 sequence and the sequence represented by SEQ ID NOs:1 and 2, the gep103 genes and polypeptides encompassed by the invention are able to complement for the lack of gep103 function (e.g., in a temperature-sensitive mutant) in a standard 35 complementation assay. Additional gep103 genes that are identified and cloned from additional Streptococcus strains, and pathogenic strains in particular, can be used to produce gep103 polypeptides for use in the various methods described herein, e.g., for identifying antibacterial agents. Likewise, the terms gep1119, gep1122, gep1315, gep1493, gep1507, gep1511, gep1518, gep1546, gep1551, gep1561, gep1580, gep1713, gep222, gep2283, gep273, gep286, gep311, gep3262, gep3387, gep47, gep61, and gep76 encompass homologs, conservative variations, and degenerate variants of the sequences depicted in FIGS. 2A-23B, respectively. Such homologs, conservative variations, and degenerate variants also are included within the invention.
Since the various GEP genes described herein have been identified and shown to be located within operons that are essential for survival, the GEP genes and polypeptides encoded by nucleic acid sequences located within operons containing GEP genes and their homologs and orthologs can be used to identify antibacterial agents. More specifically, the polypeptides encoded by nucleic acid sequences located within operons containing GEP genes can be used, separately or together, in assays to identify test compounds that bind to these polypeptides. Such test compounds are expected to be antibacterial agents, in contrast to compounds that do not bind to these GEP polypeptides. As described herein, any of a variety of art-known methods can be used to assay for binding of test compounds to the polypeptides. The invention includes, for example, a method for identifying an antibacterial agent where the method entails: (a) contacting a polypeptide encoded by a nucleic acid sequence located within an operon containing a GEP gene, or homolog or ortholog thereof, with a test compound; (b) detecting binding of the test compound to the polypeptide or homolog or ortholog; and (c) determining whether a test compound that binds to the polypeptide or homolog or ortholog inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of the test compound that binds to the polypeptide or homolog or ortholog, as an indication that the test compound is an antibacterial agent.
In various embodiments, the GEP polypeptide is derived from a non-pathogenic or pathogenic Streptococcus strain, such as Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus endocarditis, Streptococcus faecium, Streptococcus sangus, Streptococcus viridans, and Streptococcus hemolyticus. Suitable orthologs of the Streptococcus GEP genes can be derived from the bacterium Bacillus subtilis. The test compound can be immobilized on a substrate, and binding of the test compound to the polypeptide or homolog or ortholog can be detected as immobilization of the polypeptide or homolog or ortholog on the immobilized test compound, e.g., in an immunoassay with an antibody that specifically binds to the polypeptide.
If desired, the test compound can be a test polypeptide (e.g., a polypeptide having a random or predetermined amino acid sequence; or a naturally-occurring or synthetic polypeptide). Alternatively, the test compound can be a nucleic acid, such as a DNA or RNA molecule. In addition, small organic molecules can be tested. The test compound can be a naturally-occurring compound or it can be synthetically produced, if desired. Synthetic libraries, chemical libraries, and the like can be screened to identify compounds that bind to the polypeptides. More generally, binding of test compounds to the polypeptide or homolog or ortholog can be detected either in vitro or in vivo. Regardless of the source of the test compound, the polypeptides described herein can be used to identify compounds that are bactericidal or bacteriostatic to a variety of pathogenic or non-pathogenic strains.
In an exemplary method, binding of a test compound to a polypeptide encoded by a nucleic acid located within an operon containing a GEP gene can be detected in a conventional two-hybrid system for detecting protein/protein interactions (e.g., in yeast or mammalian cells). Generally, in such a method, (a) the polypeptide encoded by a nucleic acid located within an operon containing a GEP gene is provided as a fusion protein that includes the polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; (b) the test polypeptide is provided as a fusion protein that includes the test polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; and (c) binding of the test polypeptide to the polypeptide is detected as reconstitution of a transcription factor. Homologs and orthologs of the GEP polypeptides can be used in similar methods. Reconstitution of the transcription factor can be detected, for example, by detecting transcription of a gene that is operably linked to a DNA sequence bound by the DNA-binding domain of the reconstituted transcription factor (See, for example, White, 1996, Proc. Natl. Acad. Sci. 93:10001-10003 and references cited therein and Vidal et al., 1996, Proc. Natl. Acad. Sci. 93:10315-10320).
In an alternative method, an isolated operon containing a nucleic acid molecule encoding a GEP polypeptide is used to identify a compound that decreases the expression of a GEP polypeptide in vivo. Such compounds can be used as antibacterial agents. To discover such compounds, cells that express a GEP polypeptide are cultured, exposed to a test compound (or a mixture of test compounds), and the level of expression or activity is compared with the level of GEP polypeptide expression or activity in cells that are otherwise identical but that have not been exposed to the test compound(s). Many standard quantitative assays of gene expression can be utilized in this aspect of the invention.
To identify compounds that modulate expression of a GEP polypeptide (or homologous or orthologous sequence), the test compound(s) can be added at varying concentrations to the culture medium of cells that express a GEP polypeptide (or homolog or ortholog), as described herein. Such test compounds can include small molecules (typically, non-protein, non-polysaccharide chemical entities), polypeptides, and nucleic acids. The expression of the GEP polypeptide is then measured, for example, by Northern blot PCR analysis or RNAse protection analyses using a nucleic acid molecule of the invention as a probe. The level of expression in the presence of the test molecule, compared with the level of expression in its absence, will indicate whether or not the test molecule alters the expression of the GEP polypeptide. Because the GEP polypeptides are expressed from operons that are essential for survival, test compounds that inhibit the expression and/or function of the GEP polypeptide will inhibit growth of the cells or kill the cells.
Compounds that modulate the expression of the polypeptides of the invention can be identified by carrying out the assays described herein and then measuring the levels of the GEP polypeptides expressed in the cells, e.g., by performing a Western blot analysis using antibodies that bind to a GEP polypeptide.
The invention further features methods of identifying from a large group of mutants those strains that have conditional lethal mutations. In general, the gene and corresponding gene product are subsequently identified, although the strains themselves can be used in screening or diagnostic assays. The mechanism(s) of action for the identified genes and gene products provide a rational basis for the design of antibacterial therapeutic agents. These antibacterial agents reduce the action of the gene product in a wild type strain, and therefore are useful in treating a subject with that type, or a similarly susceptible type of infection by administering the agent to the subject in a pharmaceutically effective amount. Reduction in the action of the gene product includes competitive inhibition of the gene product for the active site of an enzyme or receptor; non-competitive inhibition; disrupting an intracellular cascade path which requires the gene product; binding to the gene product itself, before or after post-translational processing; and acting as a gene product mimetic, thereby down-regulating the activity. Therapeutic agents include monoclonal antibodies raised against the gene product.
Furthermore, the presence of the gene sequence in certain cells (e.g., a pathogenic bacterium of the same genus or similar species), and the absence or divergence of the sequence in host cells can be determined, if desired. Therapeutic agents directed toward genes or gene products that are not present in the host have several advantages, including fewer side effects, and lower overall dosage.
The invention includes pharmaceutical formulations that include a pharmaceutically acceptable excipient and an antibacterial agent identified using the methods described herein. In particular, the invention includes pharmaceutical formulations that contain antibacterial agents that inhibit the growth of, or kill, pathogenic Streptococcus strains. Such pharmaceutical formulations can be used for treating a Streptococcus infection in an organism. Such a method entails administering to the organism a therapeutically effective amount of the pharmaceutical formulation. In particular, such pharmaceutical formulations can be used to treat streptococcal pneumonia in mammals such as humans and domesticated mammals (e.g., cows, pigs, dogs, and cats), and in plants. The efficacy of such antibacterial agents in humans can be estimated in an animal model system well known to those of skill in the art (e.g., mouse and rabbit model systems).
Also included within the invention are polyclonal and monoclonal antibodies that specifically bind to the various GEP polypeptides described herein (e.g., gep103). Such antibodies can facilitate detection of GEP polypeptides in various Streptococcus strains. These antibodies also are useful for detecting binding of a test compound to GEP polypeptides (e.g., using the assays described herein). In addition, monoclonal antibodies that bind to GEP polypeptides are themselves adequate antibacterial agents when administered to a mammal, as such monoclonal antibodies are expected to impede one or more functions of GEP polypeptides.
As used herein, xe2x80x9cnucleic acidsxe2x80x9d encompass both RNA and DNA, including genomic DNA and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5xe2x80x2 non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence. The term xe2x80x9cisolatedxe2x80x9d can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an xe2x80x9cisolated nucleic acid fragmentxe2x80x9d is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. As used herein, the term xe2x80x9cisolated nucleic acid moleculexe2x80x9d includes an operon containing a contiguous cluster of linked sequences. xe2x80x9cIsolated operonsxe2x80x9d are those operons that are not naturally occurring and which are not associated with the sequences by which they are normally surrounded in a bacterial genome.
A nucleic acid sequence that is xe2x80x9csubstantially identicalxe2x80x9d to a GEP nucleotide sequence is at least 80% (e.g., 85%) identical to the nucleotide sequence of the nucleic acid sequences represented by the SEQ ID NOs listed in Table 1, as depicted in FIGS. 1-23B. For purposes of comparison of nucleic acids, the length of the reference nucleic acid sequence will generally be at least 40 nucleotides, e.g., at least 60 nucleotides or more nucleotides. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
The GEP polypeptides useful in practicing the invention include, but are not limited to, recombinant polypeptides and natural polypeptides. Also useful in the invention are nucleic acid sequences that encode forms of GEP polypeptides in which naturally occurring amino acid sequences are altered or deleted. Preferred nucleic acids encode polypeptides that are soluble under normal physiological conditions. Also within the invention are nucleic acids encoding fusion proteins in which a portion of a GEP polypeptide is fused to an unrelated polypeptide (e.g., a marker polypeptide or a fusion partner) to create a fusion protein. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed polypeptides, or to a hemagglutinin tag to facilitate purification of polypeptides expressed in eukaryotic cells. The invention also includes, for example, isolated polypeptides (and the nucleic acids that encode these polypeptides) that include a first portion and a second portion; the first portion includes, e.g., a GEP polypeptide, and the second portion includes an immunoglobulin constant (Fc) region or a detectable marker.
The fusion partner can be, for example, a polypeptide which facilitates secretion, e.g., a secretory sequence. Such a fused polypeptide is typically referred to as a preprotein. The secretory sequence can be cleaved by the host cell to form the mature protein. Also within the invention are nucleic acids that encode a GEP polypeptide fused to a polypeptide sequence to produce an inactive preprotein. Preproteins can be converted into the active form of the protein by removal of the inactivating sequence.
The invention also includes nucleic acids that hybridize, e.g., under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequences represented by the SEQ ID NOs. listed in Table 1, or their complements. The hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e.g., 20, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding a GEP polypeptide or its complement. Hybridizing nucleic acids of the type described herein can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Nucleic acids that hybridize to the nucleotide sequences represented by the SEQ ID NOs. listed in Table 1 are considered xe2x80x9cantisense oligonucleotides.xe2x80x9d Also included within the invention are ribozymes that inhibit the function of operons containing the GEP genes of the invention, as determined, for example, in a complementation assay.
Also useful in the invention are various cells, e.g., transformed host cells, that contain a GEP nucleic acid described herein. A xe2x80x9ctransformed cellxe2x80x9d is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding a GEP polypeptide. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, Streptococcus, Bacillus, and the like.
Also useful in the invention are genetic constructs (e.g., vectors and plasmids) that include a nucleic acid of the invention which is operably linked to a transcription and/or translation sequence to enable expression, e.g., expression vectors. By xe2x80x9coperably linkedxe2x80x9d is meant that a selected nucleic acid, e.g., a DNA molecule encoding a GEP polypeptide, is positioned adjacent to one or more sequence elements, e.g., a promoter, which directs transcription and/or translation of the sequence such that the sequence elements can control transcription and/or translation of the selected nucleic acid.
The invention also features purified or isolated polypeptides encoded by nucleic acids located within operons containing GEP genes, as listed in Table 1. As used herein, both xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d mean any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Thus, the terms gep103 polypeptide, gep1119 polypeptide, gep1122 polypeptide, gep1315 polypeptide, gep1493 polypeptide, gep1507 polypeptide, gep1511 polypeptide, gep1518 polypeptide, gep1546 polypeptide, gep1551 polypeptide, gep1561 polypeptide, gep1580 polypeptide, gep1713 polypeptide, gep222 polypeptide, gep2283 polypeptide, gep273 polypeptide, gep286 polypeptide, gep311 polypeptide, gep3262 polypeptide, gep3387 polypeptide, gep47 polypeptide, gep61 polypeptide, and gep76 polypeptide include full-length, naturally occurring gep103, gep1119, gep1122, gep1315, gep1493, gep1507, gep1511, gep1518, gep1546, gep1551, gep1561, gep1580, gep1713, gep222, gep2283, gep273, gep286, gep311, gep3262, gep3387, gep47, gep61, and gep76 proteins, respectively, as well as recombinantly or synthetically produced polypeptides that correspond to the full-length, naturally occurring proteins, or to a portion of the naturally occurring or synthetic polypeptide.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cisolatedxe2x80x9d compound is a composition that is at least 60% by weight the compound of interest, e.g., a GEP polypeptide or antibody. Preferably the preparation is at least 75% (e.g., at least 90% or 99%) by weight the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Preferred GEP polypeptides include a sequence substantially identical to all or a portion of a naturally occurring GEP polypeptide, e.g., including all or a portion of the sequences shown in FIGS. 1-23B. Polypeptides xe2x80x9csubstantially identicalxe2x80x9d to the GEP polypeptide sequences described herein have an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 99%) identical to the amino acid sequence of the GEP polypeptides represented by the SEQ ID NOs. listed in Table 1. For purposes of comparison, the length of the reference GEP polypeptide sequence will generally be at least 16 amino acids, e.g., at least 20 or 25 amino acids.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference polypeptide. Thus, a polypeptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It also might be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, other polypeptides also will meet the same criteria.
The invention also features purified or isolated antibodies that specifically bind to a GEP polypeptide. By xe2x80x9cspecifically bindsxe2x80x9d is meant that an antibody recognizes and binds to a particular antigen, e.g., a GEP polypeptide, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample that naturally includes a GEP polypeptide.
In another aspect, the invention features a method for detecting a GEP polypeptide in a sample. This method includes: obtaining a sample suspected of containing a GEP polypeptide; contacting the sample with an antibody that specifically binds to a GEP polypeptide under conditions that allow the formation of complexes of an antibody and the GEP polypeptide; and detecting the complexes, if any, as an indication of the presence of a GEP polypeptide in the sample.
Also encompassed by the invention is a method of obtaining a gene related to (i.e., a functional homolog or ortholog of) a GEP gene. Such a method entails obtaining a labeled probe that includes an isolated nucleic acid which encodes all or a portion of a GEP nucleic acid, or a homolog or ortholog thereof; screening a nucleic acid fragment library with the labeled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes; isolating labeled duplexes, if any; and preparing a full-length gene sequence from the nucleic acid fragments in any labeled duplex to obtain a gene related to the GEP gene.
The invention offers several advantages. For example, the methods for identifying antibacterial agents can be configured for high throughput screening of numerous candidate antibacterial agents.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.